The light-emitting reaction of bioluminescent bacteria is catalyzed by luciferase, a heterodimeric, mixed-function oxidase which oxidizes FMNH2 and tetradecanal to produce FMN, tetradecanoic acid, H2O, and light. The intermediates of the luciferase-catalyzed reaction have been characterized and mechanisms for the reaction have been proposed; yet, very little is known about the participation of luciferase in the reaction. The primary structures of both subunits of luciferase have been elucidated from the nucleotide sequences of lux genes from four species of bacteria, but the homology is high and sheds little light on which regions of the protein contribute to the active-site. Cline and Hastings isolated and characterized thirteen luciferase mutants of Vibrio harveyi exhibiting altered kinetics. However, the alterations in only two of the altered kinetics mutants have been determined. In order to more completely characterize the active-site of luciferase the alterations in the mutants isolated by Cline and Hastings will be identified. The mutant luxA genes will be amplified out of the bacterial chromosomes by Polymerase Chain Reaction and cloned into a phagemid vector carrying a wildtype luxB gene. Single-stranded phagemid DNA will be isolated and the luxA gene sequenced using oligonucleotides that prime dideoxynucleotide sequencing reactions at 200 nucleotide intervals. To confirm the effects of primary structure alterations, the sequence will be converted back to wildtype by site-directed mutagenesis and the enzyme kinetics compared to wildtype. Finally, the participation of identified residues in the catalyzed reaction will be characterized by making other changes in the sequence at those positions by site-directed mutagenesis. The wildtype amino acid will be replaced by residues with similar characteristics and size. Enzymes that contain substitutions at positions that participate in the reaction will be affected by the changes to a much greater extent than those with substitutions that simply contribute to the structure of the active-site.